The major goal of this proposal is to analyze the structural and biochemical differences between the axonal cytoskeletons of growing and non-growing axons. As neurons mature and stop growing, the protein composition and ultrastructure of the axonal cytoskeleton change. Functional changes such as altered rates of slow axonal transport of cytoskeletal proteins (a fundamental process in axon growth), and altered stability of cytoskeletal polymers (a key factor in stabilizing mature axons) may also occur. These alterations in the axonal cytoskeleton appear to be important in establishing an axon with a stable morphology as opposed to a dynamic, growing axon. When the distal portion of an axon is damaged by trauma, stroke, radiculopathy, or metabolic or toxic neuropathy, recovery requires successful regeneration of the distal axon. In order for axonal regeneration to take place, the axon must revert from the mature, stable state and reestablish the dynamic growth state. Axonal regeneration is thus expected to require reversal of many of the maturational changes in the axonal cytoskeleton. When dorsal root ganglia from fetal rats are maintained in culture under conditions which inhibit the growth of non-neuronal cells, they extend processes with typical axonal cytoskeletons which show changes such as increasing neurofilament protein content as they age. After two weeks of rapid axon growth at 300-400 um/day, the rate of axon growth slows to 0-40 um/day. If these slowly growing mature axons are cut, they exhibit rapid growth rates during regeneration. In the proposed experiments, these cultured axons will be examined in the immature, mature and regenerating states by four techniques. Electron micrographs of the axons will be examined to determine the ultrastructural morphology of the axonal cytoskeletons. The analysis will include morphometric determinations such as neurofilament and microtubule packing densities and nearest neighbor distance. Immunofluorescence staining will be used to detect and localize a variety of cytoskeletal proteins in the axons including microtubule-associated proteins, tyrosinated, detyrosinated and acetylated tubulins and neurofilament proteins. Differential extraction of permeabilized axons will be used to separate axonal proteins into three pools: proteins which freely diffuse out of permeabilized axons, proteins released by cold and high calcium treatment to depolymerize labile microtubules, and proteins which remain closely associated with stable microtubules and neurofilaments. These extracts will then be analyzed by gel electrophoresis, western blot and ELISA to detect the presence of specific proteins. Finally, slow axonal transport of cytoskeletal proteins will be assayed by pulse-labeling techniques and compared with axonal growth rates. The proposed studies constitute the first time all of these cytoskeletal parameters will be measured in the same neurons under controlled conditions during growth, maturity and regeneration. They will provide data concerning alterations in the structure, biochemistry and functioning of the axonal cytoskeleton associated with changes in growth state without concomitant changes in axonal interactions with non-neuronal cells. We anticipate that these data will lead to new hypotheses about the mechanisms of axon growth and regeneration, and ultimately will help to design better therapies for pathologic states which damage axons.